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Tall fescue ( Festuca arundinacea  Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes ( ACT ,  TUB ,  EF1a ,  GAPDH ,  SAND ,  CACS ,  F-box ,  PEPKR1  and  TIP41 ) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of  SAND  and  TUB  or  TIP41  and  TUB  were most stably expressed in salt-treated roots or leaves. The combinations of  GAPDH  with  TIP41  or  TUB  were stable in roots and leaves under drought stress.  TIP41  and  PEPKR1  exhibited stable expression in cold-treated roots, and the combination of  F-box ,  TIP41  and  TUB  was also stable in cold-treated leaves.  CACS  and  TUB  were the two most stable reference genes in heat-stressed roots.  TIP41  combined with TUB and  ACT  was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene  FaWRKY1  using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

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Identification and Validation of Reference Genes for Quantification of Target Gene Expression with Quantitative Real-time PCR for Tall Fescue under Four Abiotic Stresses